Activation of Aurora-A Kinase by Protein Partner Binding and Phosphorylation Are Independent and Synergistic

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2011 Nov 19

PMID 22094468

Citations 57

Authors

Charlotte A Dodson

Richard Bayliss

Affiliations

Soon will be listed here.

Abstract

Protein kinases are activated by phosphorylation and by the binding of activator proteins. The interplay of these two factors is incompletely understood. We applied energetic analysis to this question and characterized the activation process of the serine/threonine kinase Aurora-A by phosphorylation and by its protein partner, targeting protein for Xenopus kinesin-like protein 2 (TPX2). We discovered that these two activators act synergistically and without a predefined order: each can individually increase the activity of Aurora-A, and the effect of both bound together is the exact sum of their individual contributions to catalysis. Unexpectedly, the unphosphorylated enzyme has catalytic activity that is increased 15-fold by the binding of TPX2 alone. The energetic contribution of phosphorylation to catalysis is 2-fold greater than that of TPX2 binding, which is independent of the phosphorylation state of the enzyme. Based on this analysis, we propose a revised, fluid model of Aurora-A activation in which the first step is a reduction in the mobility of the activation loop by either TPX2 binding or phosphorylation. Furthermore, our results suggest that unphosphorylated Aurora-A bound to the mitotic spindle by TPX2 is catalytically active and that the phosphorylation state of Aurora-A is an inaccurate surrogate for its activity. Extending this form of analysis will allow us to compare quantitatively the effects of the whole network of kinase-activating partners. Comparison with other kinases showed that kinetic characterization detects those kinases whose activation loops undergo a rearrangement upon phosphorylation and thus whose unphosphorylated state offers a distinct target for the development of Type II inhibitors.

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References

Eyers P, Erikson E, Chen L, Maller J . A novel mechanism for activation of the protein kinase Aurora A. Curr Biol. 2003; 13(8):691-7. DOI: 10.1016/s0960-9822(03)00166-0. View

Pollard J, Mortimore M . Discovery and development of aurora kinase inhibitors as anticancer agents. J Med Chem. 2009; 52(9):2629-51. DOI: 10.1021/jm8012129. View

Wilkinson A, Fersht A, Blow D, Winter G . Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry. 1983; 22(15):3581-6. DOI: 10.1021/bi00284a007. View

Zeng K, Bastos R, Barr F, Gruneberg U . Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2. J Cell Biol. 2010; 191(7):1315-32. PMC: 3010072. DOI: 10.1083/jcb.201008106. View

Scutt P, Chu M, Sloane D, Cherry M, Bignell C, Williams D . Discovery and exploitation of inhibitor-resistant aurora and polo kinase mutants for the analysis of mitotic networks. J Biol Chem. 2009; 284(23):15880-93. PMC: 2708884. DOI: 10.1074/jbc.M109.005694. View

Huse M, Kuriyan J . The conformational plasticity of protein kinases. Cell. 2002; 109(3):275-82. DOI: 10.1016/s0092-8674(02)00741-9. View

Lew J . MAP kinases and CDKs: kinetic basis for catalytic activation. Biochemistry. 2003; 42(4):849-56. DOI: 10.1021/bi0269761. View

Johnson L, Noble M, Owen D . Active and inactive protein kinases: structural basis for regulation. Cell. 1996; 85(2):149-58. DOI: 10.1016/s0092-8674(00)81092-2. View

Toya M, Terasawa M, Nagata K, Iida Y, Sugimoto A . A kinase-independent role for Aurora A in the assembly of mitotic spindle microtubules in Caenorhabditis elegans embryos. Nat Cell Biol. 2011; 13(6):708-14. DOI: 10.1038/ncb2242. View

10.

Tyson J, Chen K, Novak B . Sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell. Curr Opin Cell Biol. 2003; 15(2):221-31. DOI: 10.1016/s0955-0674(03)00017-6. View

11.

Adams J, McGlone M, Gibson R, Taylor S . Phosphorylation modulates catalytic function and regulation in the cAMP-dependent protein kinase. Biochemistry. 1995; 34(8):2447-54. DOI: 10.1021/bi00008a007. View

12.

Sloane D, Trikic M, Chu M, Lamers M, Mason C, Mueller I . Drug-resistant aurora A mutants for cellular target validation of the small molecule kinase inhibitors MLN8054 and MLN8237. ACS Chem Biol. 2010; 5(6):563-76. DOI: 10.1021/cb100053q. View

13.

Adams J, Taylor S . Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments. Biochemistry. 1992; 31(36):8516-22. DOI: 10.1021/bi00151a019. View

14.

Stevenson L, Deal M, Hagopian J, Lew J . Activation mechanism of CDK2: role of cyclin binding versus phosphorylation. Biochemistry. 2002; 41(26):8528-34. DOI: 10.1021/bi025812h. View

15.

Zhang Y, Mei Z, Wu J, Wang Z . Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states. J Biol Chem. 2008; 283(39):26591-601. PMC: 3258911. DOI: 10.1074/jbc.M801703200. View

16.

Liu Y, Gray N . Rational design of inhibitors that bind to inactive kinase conformations. Nat Chem Biol. 2006; 2(7):358-64. DOI: 10.1038/nchembio799. View

17.

Bayliss R, Sardon T, Vernos I, Conti E . Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol Cell. 2003; 12(4):851-62. DOI: 10.1016/s1097-2765(03)00392-7. View

18.

Saylor P, Hanna E, Adams J . Mutations in the activation loop tyrosine of the oncoprotein v-Fps. Biochemistry. 1999; 37(51):17875-81. DOI: 10.1021/bi981775b. View

19.

Littlepage L, Wu H, Andresson T, Deanehan J, Amundadottir L, Ruderman J . Identification of phosphorylated residues that affect the activity of the mitotic kinase Aurora-A. Proc Natl Acad Sci U S A. 2002; 99(24):15440-5. PMC: 137735. DOI: 10.1073/pnas.202606599. View

20.

Barr A, Gergely F . Aurora-A: the maker and breaker of spindle poles. J Cell Sci. 2007; 120(Pt 17):2987-96. DOI: 10.1242/jcs.013136. View